Microporous substrate for use in a disposable bioassay cartridge

ABSTRACT

A microporous substrate for detection of surface bound target analyte molecules includes a microporous substrate material having opposed surfaces and tapered micropores extending through the substrate with the micropores having wider openings on one side of the substrate compared to the other side. The micropores have bound therein analyte specific receptors complementary to the target molecules. When a liquid sample containing the target analyte molecules with optical probes attached to the target molecules is flowed through the substrate, they bind to their complementary analyte specific receptors and emit light. This substrate structure gives an increase in the collection efficiency of light emitted from optical probes when the light is detected by a light detector spaced from the side of the substrate facing the larger micropores openings compared to a light collection efficiency of light emitted from the optical probes when the micropores are straight and not tapered.

CROSS REFERENCE TO RELATED UNITED STATES PATENT APPLICATION

This patent application is a US continuation patent application of U.S.Ser. No. 15/902,030 filed on Feb. 22, 2018, entitled DISPOSABLE BIOASSAYCARTRIDGE AND METHOD OF PERFORMING MULTIPLE ASSAY STEPS AND FLUIDTRANSFER WITHIN THE CARTRIDGE, incorporated herein in its entirety byreference, which is a US divisional patent application of U.S. Ser. No.15/564,791, filed on Oct. 6, 2017, which is a National Stage entry ofinternational application no. PCT/CA2016/050414, filed on Apr. 11, 2016.

FIELD

The present disclosure relates to a microporous substrate with taperedmicropores extending right the substrate with the openings on one sideof the substrate being wider than openings on the opposed substratesurface. The microporous substrate is for use with a disposablecartridge and method to move fluids and carry out multiple bioassaysteps within the cartridge.

BACKGROUND

Typical cartridge devices for biological assays are interfaced with aninstrument containing syringes or other types of positive displacementpumps in order to accurately meter liquid volumes required sequentiallyin a reaction zone within the disposable cartridge. This often alsoinvolves the integration of mechanical valves within the cartridgestructure to control fluid flows. In addition, care must be taken in thedesign of the fluidic paths to eliminate the formation of air bubblesthat can significantly interfere with accurate fluid transfer. Complexstructures or bubble control mechanisms are introduced into the designto mitigate these issues. This introduces manufacturing complexity andincreased cost of the cartridges which are often meant to be used in adisposable fashion.

In view of the trend toward point of use diagnostic testing, there is aneed to integrate multiple functions/assay steps in a single cartridgeon a cost effective basis consistent with mass production of thedisposable cartridges. Therefore, it would be very beneficial to providea disposable cartridge which integrates multiple functions with aminimum number of moving parts such as active pumps and valves in thefield of automated point of use diagnostic bioassays.

SUMMARY

The present disclosure is directed to a device and method to transferliquid volumes sequentially to a reaction zone with only the use ofapplied pressure or vacuum and does not require any internal valves.Fluidic transfer is limited within the cartridge by capillary pressures.Flow between reaction zones may be effected by switching pressure orvacuum between ports with external valves and hence selectivelyexceeding the capillary pressure in the elements of the cartridgeconnecting reaction zones. The pressure/vacuum source and valves arelocated in the instrument itself and are isolated from reaction fluids.None of these components are part of the disposable cartridge,significantly lowering complexity and cost.

In an embodiment there is provided a microporous substrate for use in aflow through system for detection of target analyte molecules present ina liquid sample, the target analyte molecules having optical probesbound thereto, comprising:

a microporous substrate material having opposed surfaces and taperedmicropores extending completely through a thickness of the microporoussubstrate, and wherein ends of the tapered micropores have largermicropore openings on one surface of the microporous substrate comparedto smaller pore openings on the opposed surface;

analyte-specific receptors complementary to the target analyte moleculesbeing bound to walls of the tapered micropores such that upon flowingthe liquid sample through the microporous substrate the target analytemolecules bind to the analyte-specific receptors and the optical probesemit light when the target analyte molecules in the liquid sample bindto the analyte specific receptors;

the microporous substrate being characterized such that when themicroporous substrate is spaced from the light detector with the lightdetector facing the surface of the microporous substrate having thelarger micropore openings a collection efficiency of light emitted fromanalyte-specific receptors in the tapered micropores detected by thelight detector is increased compared to a light collection efficiency oflight emitted from the analyte-specific receptors when the microporesare straight and not tapered.

The present disclosure also provides a microporous substrate fordetection of surface bound target analyte molecules, comprising:

a microporous substrate material having opposed surfaces and micropores,the micropores having bound therein analyte specific receptorscomplementary to the target analyte molecules, the pores having taperedwalls extending through a thickness of the microporous substrate inwhich the pores are wider near one surface of the substrate compared toa width of the micropores on the opposed surface to increase thecollection efficiency of light emitted from optical probes bound totarget analyte molecules when the target analyte molecules are capturedby the analyte specific receptors which is detected by a light detectorspaced from the side of the microporous substrate facing the largermicropores openings compared to a light collection efficiency of lightemitted from the optical probes when the micropores are straight and nottapered.

The present disclosure provides a method for detection of target analytemolecules, comprising:

mixing a liquid sample with a solution containing optical probes knownto bind to the target analyte molecules to form a sample solution;

flowing the sample solution through a microporous substrate materialhaving opposed surfaces and pores, the pores having bound thereinanalyte specific receptors complementary to the target analyte moleculesbeing tested for, wherein upon binding of the target analyte moleculesto the analyte specific receptors the optical probes emit light; and

the pores having tapered walls extending through a thickness of thesubstrate in which the pores are wider near one surface of the substratecompared to a width of the pores on the opposed surface to increase thecollection efficiency of light emitted from optical probes which isdetected by a light detector spaced from the side of the substratefacing the larger pore openings compared to a light collectionefficiency of light emitted from the optical probes when the pores arestraight and not tapered.

The optical probes may be selected to emit any one of colorimetriclight, fluorescent light, chemiluminescent light or bioluminescentlight.

The micropores are progressively wider near one surface of themicroporous substrate.

The micropores may have any one of a rectangular cross section, a squarecross section and a circular cross section.

The tapering of the micropores may be conical, spherical, or parabolic.

The micropores may be of uniform dimensions and morphology.

The microporous substrate may further comprise reinforcement ribs toprovide structural stability. These reinforcement ribs may be anintegral part of the microporous substrate. Alternatively, thereinforcement ribs may be separate from the substrate and made in a formof a rigid supporting mesh.

The microporous substrate may be attached to a supporting mesh placed onthe surface of the the substrate opposite to the surface with the largermicropore openings.

The microporous substrate may have a thickness of between about 0.15 toabout 0.75 mm.

The microporous substrate may be made of silicon.

The device, method disclosed herein is of particular use in the area ofmedical diagnostics (human and veterinary), food safety testing,monitoring of environmental and biological hazards and generalmeasurement of biological species. The design can be adapted to carryout most common assay formats for both proteins and nucleic acidsincluding sample preparation steps.

A further understanding of the functional and advantageous aspects ofthe present disclosure can be realized by reference to the followingdetailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will now be described, by way of example only, withreference to the drawings, in which:

FIG. 1 is a side elevational view of a pneumatically driven assaycartridge showing the core components.

FIG. 2 shows a more detailed side view of the cartridge of FIG. 1 withliquids in the starting position.

FIG. 3 is an enlarged view of the side view of FIG. 2 showing movementof the liquid (thick dark line within the capillary) from a reagentreservoir to an upper processing chamber under pneumatic control.

FIG. 4 is similar to FIG. 3 showing movement of the liquid from theupper processing chamber to a lower processing chamber through amicroporous substrate under pneumatic control.

FIG. 5 shows the liquid in the cartridge being moved back into the upperprocessing chamber under pneumatic control.

FIG. 6A shows the liquid in the cartridge partially moved into a wastecontainer after completion of the processing steps.

FIG. 6B shows a kit including a disposable cartridge along with adedicated blister pack containing a plurality of assay reagents and amatching gasket with the packets containing the assay reagents beingaligned with preselected reagent chambers.

FIG. 7 is a photograph of an assembled cartridge showing five (5)reagent/sample chambers and a bulk reagent chamber connected to acentral upper processing chamber.

FIG. 8 shows a top view of a cartridge configured for both nucleic acidsample preparation and nucleic acid amplification (isothermal orpolymerase chain reaction (PCR)) and multiplex detection of theproducts.

FIG. 9 shows a partially disassembled view of the disposable cartridgesandwiched between an upper pneumatic block assembly interface and alower thermal control assembly which form part of the instrument intowhich the cartridge is inserted.

FIG. 10 is a partial cross-sectional view of the sandwiched structure ofFIG. 9 showing a detector positioned to view the microporous substrate.

FIGS. 11A to 11C show three (3) tapered pores with different angles oftapering. Smaller tapering angle (B compared to A) leads to deepertapering. For small enough angles the tapering is continuous from onesurface of the substrate to the other as shown in C.

FIGS. 12A and 12B show optical microphotographs of the front and backsurfaces of a silicon substrate with tapered pores according to thepresent disclosure. These optical micrographs show that the highporosity of the substrate on the side with widened pores (FIG. 12A) andthe lower porosity of the substrate on the opposite side (FIG. 12B).

FIGS. 13A to 13C show micro photographs of the substrates with pores ofa different cross section with FIG. 13A being circular, FIG. 13B beingsquare, and FIG. 13C being polygonal.

FIGS. 14A to 14C show the tapered pores with different angles oftapering and as a result with different depths of tapered portion of apore, with the optical micrographs showing the cross sections of taperedpores with different angles of tapering 14A, 14B and the top view, 14Cof the substrate cross section of which is shown in FIG. 14A.

FIGS. 15A and 15B demonstrate the improvement in light transmission of amicroporous substrate due to pore tapering. The same substrate is shownin FIGS. 15A and 15B when illuminated by the same diffuse light source.The widened part of the pores are facing the objective lens in FIG. 15A,and the narrow part of pores are facing the objective lens in FIG. 15B.The spots on the substrates are regions in which the pores of thesubstrate have been blocked with probe solutions that have dried in thepores.

FIGS. 16A to 16C illustrate the mechanisms contributing to lightcollection improvement, with 16A showing the effect of increasing of theeffective depth; 16B showing the effect of an increase in the collectionangle; and 16C showing the effect of increase of surface area.

FIG. 17 shows the results of calculation of light collection efficiencyas a function of pore depth for a straight 8 um (micrometers) pore (plotA) and a pore with tapered walls (plot B).

FIG. 18 shows the results of experimental comparison of signalintensities measured with a substrate with straight pores and withtapered pores. FIG. 18 confirms the expected 40% improvement of lightcollection efficiency according to disclosure.

FIG. 19A shows another embodiment of the flow-through chip substratewith cylindrical pores with conical tapering.

FIG. 19B shows a section of a single pore of the embodiment of FIG. 19A.

FIG. 20A shows another embodiment of the flow-through chip substratewith cylindrical pores with spherical tapering.

FIG. 20B shows a section of a single pore of the embodiment of FIG. 20A.

FIG. 21A shows another embodiment of the flow-through chip substratewith cylindrical pores with parabolic tapering.

FIG. 21B shows a section of a single pore of the embodiment of FIG. 21A.

FIG. 22A shows a first embodiment of an arrangement of the taperedcylindrical pores in the microporous substrate.

FIG. 22B shows a second embodiment of tapered cylindrical pores in themicroporous substrate being more closely packed than the arrangement ofFIG. 22A with enhanced light collection efficiency.

FIG. 23 shows an embodiment of the flow-through chip substrate with thehigh-efficiency microporous substrate on the left hand side reinforcedby a frame for structural stability, shown on the right hand side of thefigure.

FIG. 24 shows another embodiment of a flow-through chip substrate forimproved optical detection sensitivity with a high-efficiencymicroporous substrate reinforced by two frames placed on the oppositesides of the substrate for structural stability.

FIG. 25A shows results of a nucleic acid bioassay conducted using theassembled cartridge shown in FIG. 7.

FIG. 25B shows the chemiluminescent image of the microporous substratecontained within the assembled cartridge shown in FIG. 7 at theconclusion of the nucleic acid bioassay

FIG. 26 shows results of a protein bioassay conducted using theassembled cartridge shown in FIG. 7.

FIG. 27A shows results of a sample preparation using a microporoussubstrate forming part of the present cartridge.

FIG. 27B shows the chemiluminescent image of a microporous substrateused to detect residual protein analytes in a solution processed by aseparate microporous substrate configured for sample preparation.

FIG. 27C shows the chemiluminescent image of a microporous substrateused to detect residual protein analytes in a solution prior toprocessing by a separate microporous substrate configured for samplepreparation.

DETAILED DESCRIPTION

Various embodiments and aspects of the disclosure will be described withreference to details discussed below. The following description anddrawings are illustrative of the disclosure and are not to be construedas limiting the disclosure. Numerous specific details are described toprovide a thorough understanding of various embodiments of the presentdisclosure. However, in certain instances, well-known or conventionaldetails are not described in order to provide a concise discussion ofembodiments of the present disclosure.

As used herein, the terms, “comprises” and “comprising” are to beconstrued as being inclusive and open ended, and not exclusive.Specifically, when used in the specification and claims, the terms,“comprises” and “comprising” and variations thereof mean the specifiedfeatures, steps or components are included. These terms are not to beinterpreted to exclude the presence of other features, steps orcomponents.

As used herein, the term “exemplary” means “serving as an example,instance, or illustration,” and should not be construed as preferred oradvantageous over other configurations disclosed herein.

As used herein, the terms “about” and “approximately” are meant to covervariations that may exist in the upper and lower limits of the ranges ofvalues, such as variations in properties, parameters, and dimensions. Inone non-limiting example, the terms “about” and “approximately” meanplus or minus 10 percent or less.

Unless defined otherwise, all technical and scientific terms used hereinare intended to have the same meaning as commonly understood to one ofordinary skill in the art.

Referring to FIG. 1, there is shown a cartridge 100 configured tofacilitate movement of fluids without the need for any internal valvesor metering devices. The design is amenable to injection moldedmanufacturing lowering cost for large volume manufacturing. Cartridge100 includes a first reagent chamber 10 which holds a liquid reagent orsample, and a second reagent chamber 12 which holds a second liquidreagent.

An upper processing chamber 14 is provided having a volume greater thanthe first reagent chamber 10 or second reagent chamber 12. Cartridge 100includes a lower processing chamber 16 which has a volume equal to orexceeding the maximum liquid capacity of upper processing chamber 14 andis designed to minimize the space between the bottom inner surface ofchamber 16 and the bottom surface of a microporous substrate 18 locatedwithin chamber 16. Cartridge 100 includes an outlet chamber 20 with avolume greater than all of the reagents and samples combined.

First reaction chamber 10 includes a pneumatic port 26 which isconfigured to provide negative differential pressure, positivedifferential pressure or vent under external system control to chamber10. Upper processing chamber 14 includes a pneumatic port 28 which isconfigured to provide negative differential pressure, positivedifferential pressure or vent under external system control to upperprocessing chamber 14. Second reaction chamber 12 includes a pneumaticport 30 which is configured to provide negative differential pressure,positive differential pressure or vent under external system control tochamber 12. Lower processing chamber 16 includes a pneumatic port 34which is configured to provide negative differential pressure, positivedifferential pressure or vent under external system control to lowerprocessing chamber 16. Similarly, outlet chamber 20 includes a pneumaticport 36 configured to provide negative differential pressure, positivedifferential pressure or vent under external system control to outletchamber 20.

Pneumatic ports 26, 28, 30, 34 and 36 may incorporate flexiblediaphragms in their respective pneumatic conduits which can be used toisolate a given chamber from a pneumatic source while allowing a flux ofgas through the conduit which is limited by the deformation of thediaphragm. Upon application of pneumatic pressure, gas will flow throughthe conduit until the back-pressure of the diaphragm equals the appliedpneumatic pressure. Such flexible diaphragms are disclosed in U.S. Pat.No. 7,470,546, which is incorporated herein by reference in itsentirety.

More particularly, flexible diaphragms may be incorporated intopneumatic ports 28 and 34 in FIG. 1 so that when a positive pneumaticpressure is applied to port 28 gas flows into upper processing chamber14 until the diaphragm deforms enough to create a back pressure equal tothe applied pressure. The gas entering the upper processing chamber 14causes the liquid to flow through the microporous substrate 18 intolower processing chamber 16, and air flows through port 34 and deformsthe diaphragm in port 34 which would be vented to the atmosphere.Although port 34 is vented to the atmosphere there would be no passageof material between the interior of the cartridge 100 and theenvironment. This configuration permits back-and-forth transport ofliquid across the microporous substrate 18 by the periodic applicationof pressure to port 28 which can be vented to the atmosphere whenpressure is not applied.

The microporous substrate 18 serves as an interface between processingchambers 14 and 16 and has a size and shape configured to prevent fluidfrom passing between processing chambers 14 and 16 other than throughthe microporous substrate 18 when the critical pressure is exceeded.Head spaces 22 are produced in lower processing chamber 16 due tomicroporous substrate 18 projecting into lower processing chamber 16.While FIG. 1 shows two (2) head spaces 22 it will be understood that thecartridge may be configured to have only one. The direction of flowdepends on the sign of the differential pressure between chambers 14 and16.

Lower processing chamber 16 includes an optical window 40 which formspart of the lower surface of this lower processing chamber 16 to allowimaging of the microporous substrate 18 from outside the devicecartridge 100. In those embodiments using microporous substrate 18 whichhas been functionalized with binding agents and which imaging is to beperformed through optical window 40, microporous substrate 18 is a rigidsubstrate disposed in a rigid plane parallel to the image plane of theimaging device such that it does not move or is not displaced whichwould result in poor quality images being detected. Preferred propertiesand structure of rigid microporous substrate 18 will be discussedhereinafter.

Upper process chamber 14 includes a solid support zone 44 which is thespace immediately above the microporous substrate 18 which can beoccupied by a solid support material of a larger size than the pores inthe microporous substrate 18 such that the material is retained in zone44 since it cannot pass through the microporous substrate 18. Thesupport material is capable of binding analytes of interest or acting asa support for reactions between bound and soluble materials.

A capillary flow channel 48 connects reagent chamber 10 with the upperprocessing chamber 14 and is designed with an inner diameter sized toprevent flow in either direction until a differential pressure isapplied exceeding a preselected critical level to permit flow betweenthe chambers 10 and 14. A capillary flow channel 50 connects reagentchamber 12 with the upper processing chamber 14 and is designed with aninner diameter sized to prevent flow in either direction until apreselected differential pressure is applied exceeding the criticallevel to permit flow between the chambers 12 and 14. A capillary flowchannel 52 connects lower processing chamber 16 with the outlet chamber20 and is designed with an inner diameter sized to prevent flow ineither direction until a preselected differential pressure is appliedexceeding the critical level to permit flow. For example, the capillaryinner diameter could be selected from the range of 50 to 500 microns toprovide critical pressures of 0.1 to 0.5 psi.

Flow is effected from one chamber to the next by applying pressure tothe originating chamber containing the fluid through the pneumatic portmounted on that chamber while simultaneously venting the destinationchamber to which the capillary channel is connected through thepneumatic port mounted on that chamber. Alternatively, negativedifferential pressure can be applied to the destination chamber whilesimultaneously venting the originating chamber. In both cases asufficient pressure differential must be provided to overcome theresistance of the channel and allow flow to occur.

In the case when a cycling of the fluid is required between two reagentchambers (e.g., for mixing) the differential pressure between thesechambers can be changed from positive to negative and back to positive.This will change the direction of fluid flow.

Reagent chambers 10 and 12 may contain liquid reagents or dried reagentsfor dissolution in the device by transferring a solution from anotherchamber. One or more of the reagent chambers 10 and 12 may be designedto accept the introduction of a sample or other material from anexternal source. It is noted that while only two (2) reagent chambers 10and 12 are shown connected to upper processing chamber 14, more could beincluded depending on the application at hand. Each reagent chamber 10and 12 is provided with the port 26 for chamber 10 and port 30 forchamber 12 which can be interfaced with an external pneumatic systemcapable of providing one or more of positive or negative pressures orventing to a given chamber under external control.

The upper processing chamber 14 is provided with port 28 which can alsobe interfaced with an external pneumatic system capable of providing oneor more of positive or negative pressures or venting to the chamberunder external control.

The internal diameter of each capillary channel 48, 50 and 52 isselected to only permit flow through the channel from one chamber to theother when a differential pressure exceeding the critical pressure isapplied. The length of the of the channel may be designed in the rangeof 5 to 30 mm in combination with the selected inner diameter in orderto control the time required to transfer the full reagent volume betweenchambers in 1 to 60 seconds using applied pressures in the range of 0.1to 1.5 psi. The internal diameter of each capillary channel 48, 50 and52 can be constant along the channel. Alternatively, a part of thechannel 48, 50 and 52 may have a smaller diameter (e.g., 50-500 um) andthe rest of the channel may have a larger diameter (e.g. 500 um-2 mm).This type of channels 48, 50 and 52 allow independent selection of thecritical pressure and flow rate.

The upper processing chamber 14 is sized to exceed the total volume ofreagents or sample fluids that may be transferred to the upperprocessing chamber 14 at any time. As seen in FIG. 1, capillary channel48 connecting reagent chamber 10 to upper processing chamber 14 andcapillary channel 50 connecting reagent chamber 12 to upper processingchamber 14 are positioned so that they terminate in the upper portion ofthe upper processing chamber 14 such that all are above the maximumlevel of liquid reached in the chamber. The bottom of the upperprocessing chamber 14 is composed of the microporous substrate 18connected to the body of the chamber 14 in such a way that fluids canonly exit through the bottom of the chamber 14 by passing through themicroporous substrate 18 when the differential pressure exceeds thecritical pressure.

The upper processing chamber 14 may also contain the solid support 44 inthe form of beads, particles, gels, or other similar materials that arecapable of binding materials of interest from fluids within the chamberor acting as a support for bound materials to interact with materialscontained in the fluid. These solid support materials 44 are ofsufficient size that they are retained by the microporous substrate 18and do not restrict flow through the substrate 18.

The microporous substrate 18 may also be composed of a material ormodified in such a way as to act as a solid support capable of bindingmaterials of interest from fluids that pass between the upper processingchamber 14 and the lower processing chamber 16 or acting as a supportfor bound materials to interact with materials contained in the fluid.

The microporous substrate 18 is constructed of material containing poresselected to provide a uniform resistance to flow across its entiresurface such that at a defined pressure differential across thesubstrate 18, fluids will pass through the pores but gases (e.g., air)will not. The properties of the pores are selected such that theresistance to flow will not be overcome by the weight of liquids in theupper processing chamber 14 or allow capillary action to draw fluidscompletely through the pores in substrate 18. The properties of themicroporous substrate 18 may optionally be selected to require apressure differential to initiate flow that is in the same range as thatrequired to initiate flow through capillaries 48, 50 and 52 in order tosimplify design of the external pneumatic system. Flow between the upperprocessing chamber 14 and the lower processing chamber 16 is effected byapplying pressure to the upper processing chamber 14 containing thefluid while simultaneously venting the lower processing chamber 16separated by the microporous substrate 18.

Alternatively, negative pressure can be applied to the lower chamber 16while simultaneously venting the upper chamber 14. In both cases thepressure differential must be provided in a range that is sufficient toovercome the resistance of the pores in the substrate 18 and allow flowof liquids to occur but below that required to overcome the resistanceto the flow of air through the pores. The process may be reversed toeffect flow in the opposite direction to allow repeated contact with thesubstrate 18 and any solid support 44 contained in the upper chamber 14as well as to provide efficient mixing.

The lower processing chamber 16 is provided with two or more ports 34(only one is shown in FIG. 1) which can be interfaced with an externalpneumatic system capable of providing one or more of positive ornegative pressures or venting to the chamber 16 under external control.The lower processing chamber 16 has a volume equal to or greater thanthe maximum volume of reagents or sample fluids that may be transferredfrom the chamber 16 at any time.

The base of the lower processing chamber 16 is positioned in closeproximity to the lower surface of the microporous substrate 18 whileadditional volume can be provided by extending a portion of the chamber16 above the outer walls of the upper processing chamber 14 to form aheadspace 22.

The lower surface of the lower processing chamber 16 which includes theoptically transparent window 40 which allows for imaging of the lowersurface of the microporous substrate 18 using for example a chargecoupled device (CCD) camera or other suitable optical sensor.

The lower processing chamber 16 is connected to one or more outletchambers 20 by one or more capillary channels 52 extending from thelowest point of the lower processing chamber 16 and terminating in theupper section of the outlet chamber 20 at a point above the maximumlevel of liquid to be contained in the outlet chamber 20. At least oneof these capillary channels 52 is positioned at the lowest level of thechamber 16 to allow substantially all of the liquid in the chamber 16 tobe removed through channel 52.

One outlet chamber 20 may be used for waste containment in which case itis sized with a volume greater than the sum of all the fluids that needto be transferred from the lower processing chamber 16. Another outletchamber (not shown) may be used to transfer fluids to additionaldownstream chambers for further processing, depending on the tests to beperformed.

In addition to controlling the flow of the fluid, the microporoussubstrate 18 alone or in combination with the solid support 44 may beused to bind components in the fluid, and the bound components may beseparated from the bulk fluid, washed, modified or copied, serve asbinding agents for additional components, recovered for further use orany combination of these steps by the sequential transport of at leastone fluid from a chamber on the device.

In addition to controlling the flow of the fluid, the microporoussubstrate 18 may be designed to bind different substances in the fluidat different regions of the substrate 18, substances bound at differentregions of the substrate 18 are subsequently detected and/or quantified.

A single device 100 may contain one or more processing zones (two areshown as processing chambers 14 and 16 but more could be included) whichuses it's integral microporous substrate 18 to accomplish differentfunctions including analyte capture (nucleic acid, protein, smallmolecule other biological or chemical entities), modification ofcaptured analyte (replication, extension, amplification, labeling,cleavage, hydrolysis), modification of soluble analytes throughimmobilized enzymes or catalysts, retention of solid matrix for highercapacity capture (beads, particles, gels), detection and/or quantitationof one or more captured analytes through optical imaging (colorimetric,fluorescent, chemiluminescent, bioluminescent). In all cases themicroporous substrate 18 also acts as a fluid control device necessaryto carry out these functions.

The side views of FIGS. 2 to 6 show side views of an actual cartridgeproduced using plastic in which a central plastic cartridge reagentplate 82 is sandwiched between an upper cartridge plate 80 and a lowercartridge plate 84. FIG. 7 shows a photograph of an assembled cartridgeand FIGS. 2 to 6 may be considered cross sections taken from FIG. 7.

FIGS. 2 and 3 illustrate the dispensing of a liquid reagent or sampleinto the upper processing chamber 14. The liquid reagent or sample 60 isloaded into the reagent chamber 10 prior to the assay through areagent/sample entry port 64 and then the port 64 is closed. A pressureof ˜1 psi is applied to the chamber 10 containing the liquid 60 via port26 while port 28 connected to the upper processing chamber 14 is ventedcreating a pressure differential allowing the reagent to flow throughthe reagent capillary channel 48 into upper processing chamber 14. Theliquid 60 falls to the bottom of the upper processing chamber 14 andcovers the integral microporous substrate 18. Any excess air is allowedto vent through port 28. This method of dispensing fluids is similar forall other reagent chambers used in the assay, with the exception of abulk wash buffer (not shown) which is stored in a larger reservoir andmetered through a capillary channel on a timed basis so that a precisevolume can be delivered during dispensing.

Referring to FIG. 4, to pull the fluid through the microporous substrate18, a differential pressure is created by applying pressure through port28, while venting to atmosphere through port 34. All other ports areclosed during cycling. Fluid 60 travels from the upper processingchamber 14 into the lower process chamber 16 and headspace 22. Byapplying a pressure differential above the critical pressure for liquidflow through the microporous substrate 18 while not exceeding thecritical pressure required for air flow through the microporoussubstrate 18, flow continues until all liquid 60 is drawn from the upperprocessing chamber 14 and then stops. This design ensures that no air isdrawn through, eliminating any bubbles that might interfere withprocessing or operation of the cartridge.

Referring to FIG. 5, to provide repeated contact with microporoussubstrate 18 alone or in combination with the solid support 44 and toensure efficient mixing, fluid 60 may be returned to the upperprocessing chamber 14 by reversing the process. A differential pressureis created by applying pressure to port 34 while simultaneously ventingto atmosphere through port 28. By applying a pressure differential abovethe critical pressure for liquid flow through the microporous substrate18 while not exceeding the critical pressure required for air flowthrough the microporous substrate 18, flow continues until all liquid 60is drawn from the lower processing chamber 16 back up to upper chamber14 and then stops. This principle eliminates the need for any precisevolumetric control of fluid flow and greatly simplifies control. Theprocess of cycling back and forth through the substrate 18 can berepeated as many times as required.

Referring to FIG. 6A, evacuation of the fluid from the lower processingchamber 16 is effected by applying a negative pressure through port 36on chamber 20 while venting to atmosphere through port 34. This allowsair to enter through the lower processing chamber 16 headspace 22 andliquid 60 to travel though a distal waste capillary channel 66 from oneside of lower chamber 16 coupled to a waste inlet 76 which empties intochamber 20 and a proximal waste capillary 70 coupled to a proximal wasteoutlet 72 exiting from the other side of chamber 16 coupled to a wasteoutlet 78 which empties liquid 60 into chamber 20.

FIG. 6B shows an embodiment which is a kit including a disposablecartridge 100 along with a dedicated blister pack 130 containing aplurality of packets 134 containing selected liquid assay reagents and amatching gasket 132 with the packets 134 containing the assay reagentsbeing aligned with preselected reagent chambers in plastic cartridgereagent plate 82. The assembled cartridge 100 with upper cartridge plate80 includes the packets 134 partially projecting into theircorresponding reagent chambers. When inserted into the instrument toimplement the biological assay, applying pressure via the pneumaticsystem coupled to the pneumatic ports on plate 80 (not shown) of thevarious chambers results in rupturing of frangible seals in the blisterpack resulting in the reagents flowing into their respective chambers.The gasket 132 provides a liquid and gas seal between chambers.Additional solid reagents may be deposited into preselected reagentchambers within plastic cartridge reagent plate 82 prior to assembly ofcartridge 100, providing flexibility in the customization of reagentselection for desired biological assays and simplifying storage andtransport requirements.

As noted above, FIG. 7 is a photograph of an assembled cartridge showingfive (5) reagent/sample chambers 10 connected to a central upperprocessing chamber 14. This photograph shows the cartridge without thepneumatic connection to the cartridge. A nucleic acid bioassay (FIG. 25)and a protein bioassay (FIG. 26) were conducted using the assembledcartridge shown in FIG. 7.

Analysis of nucleic acids usually requires processing steps to isolatenucleic acids and to derive labelled copies of them for subsequentdetection. Many applications require the analysis of many differenttarget sequences, and high analytical sensitivity is often required.Furthermore, automated, cost-effective systems will be required so thatrelatively unskilled people will be able to perform the tests reliablyfor routine clinical testing.

Purification and amplification of multiple nucleic acids targets can beperformed by capturing the nucleic acids on a solid support andperforming a series of incubation and washing steps on the support toproduce derivatives of the nucleic acids that can be analyzed byhybridization on nucleic acid probes arrayed on the microporoussubstrate.

FIG. 8 and its included legend shows a top view of a configuration of abioassay cartridge 200 which incorporates design of cartridge 100 but isconfigured for both nucleic acid sample preparation and nucleic acidamplification (isothermal or polymerase chain reaction (PCR)) andmultiplex detection of the products. Cartridge 200 is configured forboth sample preparation using one microporous support 18 in processingchamber 209 and reaction product detection using a second poroussubstrate 18 in processing chamber 224 each consisting of an upperprocessing chamber 14 and a lower processing chamber 16 separated bymicroporous substrate 18.

Cartridge 200 provides for a sample inlet 208, a means to mix the samplewith a lysis or pretreatment buffer 210, a processing chamber 209containing microporous substrate 18 in which capture and modification ofnucleic acids from the sample can be performed using dried or liquidreagents supplied from chambers 205, 207, 201, 202,203, 204, or 206.Fluids from the processing chamber 209 may be transferred to wastechamber 226 or in the case of fluid containing the derivative nucleicacids to a thermal treatment chamber 211 or intermediate chamber 212.

Chamber 212 may be used to mix the fluid with dried or liquid reagent inchamber 213. Subsequently, the fluid may be processed through one ormore temperature treatment chambers 214, 216 where isothermal or thermalcycling amplification may take place. These thermal treatment chambers211, 214, 216 are isolated from the bulk of the cartridge by thermalinsulating zones 215 and controlled by the application of heat orcooling from an external thermal control assembly 108 (FIG. 9). Theprocessed liquid containing the amplified derivative nucleic acids canthen be transferred to an intermediate chamber 218, mixed with anappropriate binding buffer 219 for hybridization to the microporoussubstrate 18, located in sample processing chamber 224 where thederivative nucleic acids are detected on bound nucleic acid probesimmobilized in specific locations on the microporous substrate 18.

A series of steps as previously described are carried out using reagentsfrom adjacent chambers 217, 220, 222, 223, 225 with spent fluids beingdirected to waste chamber 227. In all cases pneumatic pressure appliedthrough ports located on each chamber is used to control fluid movement.As a final step, an image of the microporous substrate 18 is capturedwith a CCD camera with integral lens 120 (FIG. 10) located below theoptical window 40 (FIG. 1). This image is analyzed for intensity oflight measured across the microporous substrate and correlated to thespecific regions known to contain the immobilized probes. Thisinformation is used to calculate the presence or absence or quantity ofspecific nucleic acids in the original sample.

Generally speaking, using the design principles disclosed above,cartridges may be configured to have multiple reagent/samplechambers/reservoirs, upper and lower processing chambers 14 and 16, andwaste chambers 20. For example, waste chamber 20 may in fact be anintermediate chamber accepting reaction products from a first processingstation including first and second upper and lower processing chambers14 and 16 with chamber 20 forming a sample chamber for a second seriesof upper and lower processing chambers 14 and 16.

It will be understood that cartridge 200 may be configured withadditional features to permit numerous intermediate processing steps tobe carried out between the first and second set of upper and lowerprocessing chambers 14 and 16. Non-limiting examples of theseintermediate processing steps may include mixing, dilution, incubation,thermal treatment including but not limited to thermal cycling to give afew examples. Optionally cartridge 200 may include a decontaminationchamber 228 containing a cleansing agent selected to destroy orneutralize harmful products of the assay or sample.

The system of FIG. 8 utilizing the disposable cartridge disclosed hereinis very amenable to performing the above noted nucleic acid assay suchas that disclosed in United States Patent Publication Serial No.US2018-0057855A1, which is incorporated herein by reference in itsentirety, and which is a national phase entry patent application ofPCT/2016/050367 filed on Mar. 29, 2016. Thus, the present disclosureprovides a cartridge which in an embodiment comprises two differentmicroporous substrates each with upper and processing chambers, one ofwhich is a solid support for purification of multiple target nucleicacids and processing of the target nucleic acids to produce derivativenucleic acids, and the other of which is a porous substrate on which thederivative nucleic acids are detected on bound nucleic acid probes. Thepresent cartridge, in conjunction with an instrument designed to operateit, will accept samples and provide clinically relevant informationwithout user intervention after inserting the samples.

Analysis of proteins in biological samples (e.g., human serum) byimmuno-binding reactions often requires dilution of the samples beforethe immuno-binding reactions. The present disclosure providesembodiments of a disposable cartridge comprising two differentmicroporous substrates 18 each with associated upper and lowerprocessing chambers 14 and 16, one of the coupled chambers 14 and 16 maybe used for mixing of the sample with a diluent, and the second of thecoupled chambers 14 and 16 includes a flow-through microporous substrate18 on which the proteins are detected by immuno-binding reactions.

Specific volumes of the sample and of the diluent are transported to theupper processing chamber 14 above the first microporous support 18, andthey are mixed by passing the solution through the porous substrate 18into the lower processing chamber 16, and are pneumatically cycled ordriven back and forth between the chambers 14 and 16 at least one timebefore the diluted samples are transported from the first lowerprocessing chamber 16 to the second buffer processing chamber 14 abovethe second microporous substrate 18 for detection on the secondmicroporous substrate 18. The first microporous substrate 18 may containimmobilized binding agents that would bind specific components in thesample. For example, interfering substances might be removed by bindingto the first microporous substrate 18 before the immuno-binding step onthe second porous substrate 18 is performed.

In another instance, low abundance substances may be concentrated from alarge volume by binding to the first microporous substrate 18 and thenbeing released in a smaller volume at higher concentration before theimmuno-binding step on the second microporous substrate 18 is performedin order to improve overall sensitivity of detection.

FIG. 9 shows a partially exploded view of the disposable cartridge 104sandwiched between an upper pneumatic block assembly interface 106 and alower thermal control assembly 108 which form part of the instrumentinto which the cartridge 104 is inserted. Pneumatic interface 106includes all the requisite pneumatic coupling components, tubes and thelike needed to couple to the pneumatic ports of the cartridge 104. Allthese components are housed in interface 106 and do not form part of thedisposable cartridge 104.

Similarly, thermal control assembly 108 contains all requisite featuressuch as heaters, temperatures sensors and associated controllers,microprocessors and the like to control the temperature in selectedzones of the cartridge 104. The thermal control assembly 108 includes acentral aperture 110 which when assembled with cartridge 104 aligns withoptical window 40 to allow imaging of the porous substrate 18. FIG. 10is a partial cross sectional view of the sandwiched structure of FIG. 9showing detector 120 positioned to view this microporous substrate 18 inthe assembled system. Detector 120 which includes an appropriateobjective lens is configured to image the bottom side of microporoussubstrate 18 to detect the presence of colorimetric, fluorescent,chemiluminescent, or bioluminescent signals.

A preferred material from which the microporous substrate 18 is producedis silicon which is rigid and opaque to chemiluminescent emission. Thisopacity prevents crosstalk between different pores of the substrate andhence prevents crosstalk between closely spaced regions on the substratewith different binding agents. This permits the analysis of manyanalytes in a small device, since different binding agents can bearranged in close proximity. As an example, the substrate may containpores with a size in the range of 1 to 15 microns with wall thicknessesbetween pores ranging from 1 to 5 microns.

Referring to FIGS. 11A to 14C inclusive, in an embodiment of themicroporous substrate 18, the two opposed sides have different poresizes. The side of the substrate 18 from which light is collected toenable detection and analysis has substantially wider pores as can beseen in FIGS. 11A to 14C, and this side is the side facing into lowerreaction chamber 16 and faces the optical window 40 from which thedetector 120 (FIG. 10) is spaced. As can be appreciated from FIGS. 11Ato C, the walls of the pores at this surface are tapered rather thanbeing normal to the surface. This geometry presents a greater surfacearea to the detection optics and less restriction to the transmission oflight from within the pores. Despite the large pores on a front surfaceand great porosity, the substrate 18 has adequate strength andstructural stability for flow-through applications due to the small poresize on the opposite side and there is a substantial amount of materialbetween the pores.

The remarkable asymmetric optical properties of the substrate areillustrated in FIGS. 15A and B. Specifically, FIGS. 15A and 15Bdemonstrate the improvement in light transmission of a porous substratedue to pore tapering. The same substrate is shown in 15A and 15B whenilluminated by the same diffuse light source. The widened part of thepores are facing the objective lens in FIG. 15A and the narrow part ofthe pores are facing the objective lens in FIG. 15B. The spots on thesubstrates are regions in which the pores of the substrate have beenblocked with probe solutions that have dried in the pores.

Tapering of the pore walls provides improvement of light collection dueto increase of the depth from which the light can be collected, increaseof the emitting surface area of the upper portion of a pore and increaseof a collection angle. These mechanisms of light collection efficiencyare illustrated in FIGS. 16A to 16C with 16A showing the effect ofincreasing of the effective depth; 16B showing the effect of an increasein the collection angle; and 16C showing the effect of increase ofsurface area.

The results of the evaluation of these effects for a particularimplementation of the method described in this application are shown inFIG. 17 which shows the results of calculation of light collectionefficiency as a function of pore depth for a straight 8 um pore (plot A)and a pore with tapered walls (plot B). The parameters used for thisevaluation are: 1) the width of non-tapered portion of a pore is 8 um;2) the thickness of a wall between pores is 4 um; 3) the substratethickness is 350 um; 4) tapering angle 2 degrees; 5) the diameter of theobjective lens is 25.4 mm; and 6) the working distance of the objectivelens is 50 mm.

In FIG. 17 the rise of the flat part of the curve is caused by increaseof the collection surface area, the shift of the curve is caused byincrease of the pore depth from which the light collection is limited bythe parameters of the optical assembly rather than the pore walls, thechange in a slope of the curve is associated with a change of thecollection angle. As a result, the expected improvement of lightcollection efficiency is 1.4 to 1.5 fold.

The substrate 18 using silicon has been used to manufacture flow-throughchips on which different probes have been immobilized in discreteregions or spots. The same flow-through chips have been manufacturedwith a highly microporous silicon substrate with pore walls normal tothe surface. When these flow-through chips were hybridized with the sametarget molecules and processed with identical protocols to detectchemiluminescent labels attached to target molecules bound by theprobes, the signal intensities were approximately 40% greater with thesubstrate described in this invention (FIG. 18). This experimentalresult confirms the theoretical evaluation of efficiency enhancement dueto pore tapering. The enhanced optical detection sensitivity improvesthe sensitivity of assays performed on the chips and/or improves thethroughput of the assay system.

The suggested approach is not very sensitive to a particular selectionof the tapering angle as long as the inner plane of a pore wall does notrestrict light collection. For the parameters listed above the taperingangle can be selected in the range between 0.3 degrees (tapering of apore wall along full pore depth) to approximately 14 degrees. Taperingwith the angles outside of this range will still increase amount ofcollected light, but the improvement will be less pronounced. It isnoted that selection of a particular tapering angle and depth oftapering can be additionally influenced by the process of substratemanufacturing, the selected pore size and membrane thickness.

The geometry of pores does not need to be square. If the manufacturingprocess requires they may have a different cross section, for example,circular. In this case the pore is cylindrical (see FIGS. 19A, 19B to21A, 21B inclusive). In this case the simplest form of tapering isconical as shown in FIGS. 19A and 19B. The light collection efficiencycan be additionally increased by changing shape of tapering from conicalto spherical (see FIGS. 20A and 20B) or parabolic (FIGS. 21A and 21B).

Pores of different cross section (circular, square, polygonal) werederived to practice: the micro photographs of such silicon substratesare shown in FIGS. 13A to 13C. The light collection efficiency can beadditionally improved for a substrate with cylindrical pores by a denserarrangement of pores as shown in FIG. 22B compared to the collectionefficiency of the packed structure of FIG. 22A.

The structural stability of the substrate material depends on the typeof material (e.g., silicon or plastic) and its thickness. If thesubstrate is thin or/and the material is flexible or soft, areinforcement frame can be used to strengthen the substrate (see FIGS.23 and 24). Referring to FIG. 23, a substrate 300 can be attached to asingle frame 310 made of ribs 304 with the single frame being integrallyformed with the substrate 300 or, preferably sandwiched between twoseparate frames 324 and 326 (see FIG. 24), which are separate from thesubstrate 300, to allow bidirectional application of pressure requiredto drive fluids through the porous substrate as described above withoutdamaging of the substrate.

in conclusion, the present disclosure provides a disposable samplehandling cartridge for performing multiplex biological assays in whichthe cartridge is designed and configured to provide complex fluidprocessing without the need for active pumping and valving. Thecartridge is readily produced using standard molding techniques, nonanostructrures are required and no precise tolerances are required. Themovement of sample and reagent fluid is solely determined by applicationof differential pressures, which are correlated primarily with theproperties of the sample substrate 18, namely pore size and distributionin the substrate 18, as well as the inner diameter of the capillarychannels (e,g. 48). The cartridge disclosed herein advantageouslycontains no moving parts and is made of a small number of parts comparedto current systems, which typically contain active pumps, active valvesand the like.

The cartridge disclosed herein may be used for, but is not limited touse in sandwich, or competitive immunoassay for protein antigenanalysis; serology for antibody binding to immobilized antigens forallergy, autoimmune, infectious disease; nucleic acids measurement ofDNA, RNA, mRNA, microRNA (miRNA) etc. to identify specific sequenceswhose presence or expression is correlated to presence or progress ofdisease, sequences that can be used to identify species of bacteria,fungi, viruses in a sample, sequences that indicated the presence ofspecific resistance genes in pathogens, measurement of copy numbervariations (CNV's) or specific gene variants or deletions that correlateto risk of disease, gene signatures used to type samples for forensic oridentification purposes. In addition, it may be used for small moleculemeasurements including drugs and environmental contaminants. It may alsobe used in multiple sample matrices including human and animal fluidsand tissues, food and agricultural samples, environmental samples, cellsand lysates of cells, and bioprocessing fluids.

Non-limiting exemplary uses of the disposable cartridge disclosed hereinwill now be given using a nucleic acid assay and a protein assay.

Examples

FIG. 25A shows results of a nucleic acid bioassay wherein a samplecontaining biotin labelled PCR products representing copies of specificgene sequences from bacterial samples were processed using the cartridgeshown in FIG. 7. Prior to assembly of the cartridge, the microporoussubstrate 18 was functionalized in discrete regions to form analysisspots, each of approximately 200 um in diameter, with oligonucleotideprobes containing sequences complementary to sequences known to occur inthe amplified bacterial gene (+ve Probes 1, 2, 3, 4), sequences notknown to occur in the amplified bacterial gene (−ve Probes 1, 2) or asequence complementary to an artificial oligonucleotide added to thesample (Fiducial). In addition, one blank spot where no oligonucleotideprobe was immobilized was used as a control to measure backgroundsignal. 5 individual reagent wells 10 and a bulk chamber 87 were used.

The reagent chambers were individually loaded with blocking buffer,hybridization buffer, sample, streptavidin-HRP and chemiluminescentsubstrate respectively. The bulk reservoir 87 was loaded with washbuffer. Reagents were transferred to the upper processing chamber inindividual steps as illustrated in FIG. 3. Each liquid was thentransferred to the lower processing chamber as illustrated in FIG. 4 andthen returned to the upper processing chamber as illustrated in FIG. 5.

After repeating this cycle back and forth through the microporoussubstrate 18 as many times as required for each step the reagent wasremoved to waste chamber as illustrated in FIG. 6. Between each step analiquot of wash buffer from bulk chamber 87 was similarly processed. Thesequential steps accomplished blocking of the microporous substrate toprevent non-specific binding, hybridization of PCR products in thesample to the probes containing complementary sequences immobilized indiscrete regions on porous substrate 18, binding of streptavidin-HRP tothe biotin label on captured PCR products, and introduction of achemiluminescent substrate that could be processed by the captured HRPenzyme to produce a chemiluminescent emission in that specific region.

During the final step, an image of the microporous substrate 18 wascaptured with a CCD camera 120 located below the optical window 40. Thisimage FIG. 25B was analyzed for intensity of light measured across themicroporous substrate 18 and correlated to the specific regions known tocontain the immobilized probes. FIG. 25A shows the luminescent intensityfor three repeats of the bioassay for the same sample. It will be notedthat significant signals are observed on analysis spots formed byimmobilizing probes containing complementary sequences to gene sequencesexpected in the sample (+ve Probes 1, 2, 3, 4), minimal signal isobserved on analysis spots formed by immobilizing probes containingcomplementary sequences to gene sequences not expected in the sample(−ve Probes 1, 2). As expected, no signal was observed on the blankanalysis spot, and substantial signal was observed on the analysis spotcontaining a complementary sequence to the artificial oligonucleotideadded to the sample prior to analysis.

FIG. 26 shows results of protein bioassays on human serum or controlbuffer to determine the presence of antibodies against the measles viruscarried out using the cartridge pictured in FIG. 7. Prior to assembly ofthe cartridge, the microporous substrate 18 was functionalized indiscrete regions to form analysis spots, each of approximately 200 um indiameter, with a deactivated measles virus preparation. Four reagentchambers were individually loaded with blocking buffer, sample, HRPlabelled anti human immunoglobulin G and chemiluminescent substrate,respectively. The bulk reservoir 87 was loaded with wash buffer.

Reagents were transferred to the upper processing chamber in individualsteps as illustrated in FIG. 3. Each liquid was then transferred to thelower processing chamber as illustrated in FIG. 4 and then returned tothe upper processing chamber as illustrated in FIG. 5. After repeatingthis cycle back and forth through the microporous substrate 18 as manytimes as required for each step the reagent was removed to waste chamberas illustrated in FIG. 6. Between each step an aliquot of wash bufferfrom bulk chamber 87 was similarly processed. The sequential stepsaccomplished blocking of the microporous substrate to preventnon-specific binding, binding from the sample of any immunoglobulinscontaining regions that are specific to components of the measles virusimmobilized in discrete regions on microporous substrate 18, binding ofanti-human immunoglobulin G antibody coupled to a HRP enzyme to anyretained anti-measles immunoglobulins, and introduction of achemiluminescent substrate that could be processed by the bound HRPenzyme to produce a chemiluminescent emission in that specific region.

During the final step, an image of the microporous substrate 18 wascaptured with a CCD camera 120 located below the optical window 40. Thisimage was analyzed for intensity of light measured across themicroporous substrate 18 and correlated to the specific regions known tocontain the immobilized virus. The chart in FIG. 26 shows theluminescence intensity recorded for three types of samples. It will benoted that significant signal corresponding to the presence of measlesspecific antibodies is observed from the serum sample drawn from apatient known to have immunity to the measles virus (positive serum).Significantly lower signal is observed from serum drawn from a patientknown to have reduced immunity to the measles virus (negative serum).Minimal signal is observed from a control buffer sample that does notcontain any measles specific antibodies.

FIG. 27A illustrates the results of a process that utilizes twodifferent microporous substrates each with upper and processingchambers, one of which is a solid support for capture of proteinanalytes, and the other of which is a microporous substrate on which theprotein analytes are detected on bound protein specific receptors. Inthis example, identical samples containing biotinylated mouse IgGanalyte were cycled through a microporous substrate 18 that wasfunctionalized with a rabbit anti-mouse antibody know to have a highbinding affinity for mouse IgG (Treated sample) or cycled through amicroporous substrate 18 that had not been functionalized, (Non-treatedsample).

The resulting fluid was then processed through a microporous substratethat had been functionalized in discrete regions to form analysis spots,each of approximately 200 um in diameter with either a rabbit anti-mouseantibody known to have a high binding affinity for mouse IgG or abiotinylated bovine serum albumin to serve as a reference spot. Washing,binding of streptavidin-HRP to any captured biotin-mouse IgG andimmobilized biotin-BSA, and introduction of a chemiluminescent substratethat could be processed by the bound HRP enzyme to produce achemiluminescent emission in that specific region were sequentiallycarried out. During the final step, an image of the microporoussubstrate 18 was captured with a CCD camera 120 located below theoptical window 40. The intensity of each spot functionalized with rabbitanti-mouse IgG correlates with the amount of biotinylated mouse IgGanalyte present in the solution.

FIG. 27A shows that the sample that had been processed by the firstfunctionalized microporous substrate 18 (Treated) was almost completelydepleted of mouse IgG analyte when processed on the second microporoussubstrate 18 used for detection. It can also be observed that the samplethat had been processed by the first non-functionalized microporoussubstrate 18 (non-treated) exhibited high levels of mouse IgG analytewhen processed on the second microporous substrate 18 used fordetection.

FIG. 27B represents the signal captured by the CCD camera 120 from thesecond microporous substrate 18 for the treated sample. Significantsignal is observed only on the reference biotinylated BSA analysisspots. FIG. 27C represents the signal captured by the CCD camera 120from the second microporous substrate 18 for the non-treated sample.Significant signal from the analysis spots for both the referencebiotinylated BSA and the biotinylated mouse IgG can be observed. Thisillustrates the high efficiency of using a first microporous substrate18 functionalized with analyte specific reagents to deplete thoseanalytes prior to detection and quantitation on a second microporoussubstrate 18. As an example, this may have utility in removing ordepleting substances that may interfere with analysis on the secondmicroporous substrate 18.

Therefore what is claimed is:
 1. A microporous substrate for use in aflow through system for detection of target analyte molecules present ina liquid sample, the target analyte molecules having optical probesbound thereto, comprising: a microporous substrate material havingopposed surfaces and tapered micropores extending completely through athickness of said microporous substrate, and wherein ends of the taperedmicropores have larger micropore openings on one surface of themicroporous substrate compared to smaller pore openings on the opposedsurface; analyte-specific receptors complementary to the target analytemolecules being bound to walls of the tapered micropores such that uponflowing the liquid sample through the microporous substrate the targetanalyte molecules bind to the analyte-specific receptors and the opticalprobes emit light when the target analyte molecules in the liquid samplebind to the analyte specific receptors; the microporous substrate beingcharacterized such that when said microporous substrate is spaced fromsaid light detector with said light detector facing the surface of saidmicroporous substrate having the larger micropore openings a collectionefficiency of light emitted from analyte-specific receptors in saidtapered micropores detected by the light detector is increased comparedto a light collection efficiency of light emitted from theanalyte-specific receptors when the micropores are straight and nottapered.
 2. The microporous substrate according to claim 1 in which thepores are progressively wider near one surface of the microporoussubstrate.
 3. The microporous substrate according to claim 1, whereinthe micropores have any one of a rectangular cross section, a squarecross section and a circular cross section.
 4. The microporous substrateaccording to claim 1, wherein tapering of the micropores is conical,spherical, or parabolic.
 5. The microporous substrate according to claim1, which the micropores are of uniform dimensions and morphology.
 6. Themicroporous substrate according to claim 1, further comprisingreinforcement ribs to provide structural stability.
 7. The microporoussubstrate according to claim 6 in which the reinforcement ribs are anintegral part of the microporous substrate.
 8. The microporous substrateaccording to claim 6, wherein the reinforcement ribs are separate fromthe substrate and made in a form of a rigid supporting mesh.
 9. Themicroporous substrate according to claim 11 with the said microporoussubstrate is attached to a supporting mesh placed on the surface of thesaid substrate opposite to the surface with the larger microporeopenings.
 10. The microporous substrate according to claim 1 having athickness of between about 0.15 to about 0.75 mm.
 11. The microporoussubstrate according to claim 1 made of silicon.
 12. A microporoussubstrate for detection of surface bound target analyte molecules,comprising: a microporous substrate material having opposed surfaces andmicropores, the micropores having bound therein analyte specificreceptors complementary to the target analyte molecules, the poreshaving tapered walls extending through a thickness of said microporoussubstrate in which the pores are wider near one surface of the substratecompared to a width of the micropores on the opposed surface to increasethe collection efficiency of light emitted from optical probes bound totarget analyte molecules when the target analyte molecules are capturedby the analyte specific receptors which is detected by a light detectorspaced from the side of the microporous substrate facing the largermicropores openings compared to a light collection efficiency of lightemitted from the optical probes when the micropores are straight and nottapered.
 13. A method for detection of target analyte molecules,comprising: mixing a liquid sample with a solution containing opticalprobes known to bind to the target analyte molecules to form a samplesolution; flowing the sample solution through a microporous substratehaving opposed surfaces and pores, the pores having bound thereinanalyte specific receptors complementary to the target analyte moleculesbeing tested for, wherein upon binding of the target analyte moleculesto the analyte specific receptors the optical probes emit light; and thepores having tapered walls extending through a thickness of saidsubstrate in which the pores are wider near one surface of the substratecompared to a width of the pores on the opposed surface to increase thecollection efficiency of light emitted from optical probes which isdetected by a light detector spaced from the side of the substratefacing the larger pore openings compared to a light collectionefficiency of light emitted from the optical probes when the pores arestraight and not tapered.
 14. The method according to claim 13, whereinthe optical probes are selected to emit any one of colorimetric light,fluorescent light, chemiluminescent light or bioluminescent light.